Neuroblastoma (NB) continues to be the most common extracranial solid tumor in children. The majority of high-risk NB patients show an initial response to therapy but ultimately relapse, suggesting that acquired drug resistance or selection of therapy-resistant cells occurs with chemotherapy treatment. This presents a major obstacle for treatment. Understanding the molecular mechanisms that mediate resistance to chemotherapy and targeting key molecules in this pathway are pivotal for curing this disease. NF-B activation is frequently encountered in tumor cells and it is believed to be one of the mechanisms of cancer chemotherapy resistance. Chemotherapeutic agents and radiation therapy can activate NF-B. TAK1 is a pivotal kinase intermediate for IKK and MAPK activations, as well as IL-6 gene expression in response to multiple stimuli. Recently, interactions between tumor and inflammatory cells have been reported to contribute to the clinical metastatic NB phenotype. A galectin-3-dependent pathway in NB cells has been found to upregulate IL-6 in the microenvironment of human NB. IL-6 expression in stromal cells and macrophages promotes NB proliferation in tumor microenvironment. TAK1 mediates NF-B and MAPK activations in response to genotoxic stresses. Given that NF-B and MAPK activations are two major survival signals, we hypothesize that inhibition of TAK1 activation may disrupt the balance between cell-death and cell-survival, and sensitize cells to chemotherapy resulting in cell death. Furthermore, TAK1 inhibition in stromal cells and tumor-associated macrophages may block tumor-induced IL-6 expression and disrupt their functional interaction with NB cells in tumor microenvironment. In our preliminary studies, we have found that TAK1 inhibition by a small molecule inhibitor (5Z-7-oxozeaenol) significantly enhances the sensitivity of NB cells to chemotherapy in vitro and in vivo. The central hypothesis of this work is that TAK1 plays an important role in chemoresistance of NB by mediating the interaction of NB tumor cells with stromal cells and tumor-associated macrophages in the tumor microenvironment. The proposed experiments will test this hypothesis by using an orthotopic and TH-MYCN transgenic mouse models to analyze the effect of TAK1 inhibitor on tumor chemoresistance and tumor microenvironment. The specific aims for this application are: 1) to determine whether TAK1 inhibitor sensitizes NB cells to chemotherapy in both orthotopic xenograft and TH-MYCN transgenic mouse models of NB; 2) to determine the effect of TAK1 inhibition on the interaction of NB cells and tumor microenvironment in these models. The proposed project, will establish TAK1 as a therapeutic target in NB. Furthermore, this small molecule inhibitor of TAK1 kinase may serve as a potential adjunct in the treatment of high-risk NB patients. The long-term goal of this proposal is to identify and validate potential druggable enzymatic targets for therapeutic intervention of this devastating disease in children.